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AMALGKIT builds RNA-seq expression datasets from public SRA records and private FASTQ files. The current CLI is Python-only: the main pipeline no longer requires R, Rscript, or R packages.

Good first pages:

• Installation and dependencies
• Tutorial 1
• Parallel processing
• v0.16.25 notes

Workflow Map

The usual public-SRA workflow is:

dataset -> metadata -> select -> getfastq -> quant -> merge -> finalize

Cross-species projects often add ortholog-aware normalization and filtering after merge:

merge + BUSCO/orthogroups -> cstmm -> wsfilter -> csfilter -> finalize

Private FASTQ projects start by integrating local files into metadata:

integrate -> getfastq -> quant -> merge -> finalize

Validation and recovery are separate maintenance steps:

sanity -> rerun

Typical Public-SRA Run

amalgkit dataset --rule_set base --out_dir ./ --overwrite yes
amalgkit metadata \
    --out_dir ./ \
    --entrez_email example@email.com \
    --search_string '("platform illumina"[Properties]) AND ("type rnaseq"[Filter])'
amalgkit select --out_dir ./
amalgkit getfastq --out_dir ./
amalgkit quant --out_dir ./ --build_index yes
amalgkit merge --out_dir ./
amalgkit finalize --out_dir ./ --batch_effect_alg no

Add BUSCO-based cross-species steps when you have compatible transcriptome FASTA files or BUSCO tables:

amalgkit busco --out_dir ./ --lineage eukaryota_odb12
amalgkit cstmm --out_dir ./ --dir_busco ./busco
amalgkit wsfilter --out_dir ./
amalgkit csfilter --out_dir ./ --metadata ./wsfilter/metadata.tsv --dir_busco ./busco
amalgkit finalize --out_dir ./ --metadata ./csfilter/metadata.tsv --batch_effect_alg latent_glm

Active Commands

Command	Role
amalgkit dataset	extract bundled datasets, initialize workspaces, and export select_rules.tsv
amalgkit metadata	retrieve SRA metadata by Entrez query or species-wise batch files
amalgkit integrate	add local FASTQ files to AMALGKIT metadata
amalgkit select	apply select_rules.tsv and mark rows for downstream processing
amalgkit getfastq	download public SRA data or stage private FASTQ files
amalgkit quant	quantify transcript abundance with kallisto or oarfish
amalgkit merge	merge per-run quantification into per-species tables
amalgkit busco	create BUSCO tables for ortholog-aware downstream steps
amalgkit cstmm	run cross-species TMM normalization using single-copy genes
amalgkit wsfilter	perform within-species outlier filtering
amalgkit csfilter	perform cross-species outlier filtering
amalgkit finalize	export final expression tables and optional batch-corrected outputs
amalgkit sanity	check expected outputs in a workspace
amalgkit rerun	rerun failed targets recorded by sanity

Legacy Command Migration

These commands existed in older AMALGKIT releases but are not part of the current CLI:

Old command	Current workflow
amalgkit config	use amalgkit dataset --rule_set ... to write select_rules.tsv, then run amalgkit select
amalgkit curate	run amalgkit wsfilter, optionally amalgkit csfilter, then amalgkit finalize
amalgkit csca	use amalgkit csfilter for cross-species filtering and amalgkit finalize for final tables